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| Home > Publications database > Untersuchungen zur Glykolyse und zum L-Serin-Stoffwechsel in Corynebacterium glutamicum |
| Dissertation / PhD Thesis/Book | PreJuSER-31347 |
2004
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
Please use a persistent id in citations: http://hdl.handle.net/2128/104
Report No.: Juel-4130
Abstract: The amino acid L-serine is a central metabolite in the metabolism and of increasing importance for the industrial use. The aim of this work was to investigate if L-serine can be produced biotech nologically by Corynebacterium glutamicum and to study the influence of L-serine degradation and the provision of glycolytic precursors. To improve the availability of glycolytic precursors for the L-serine biosynthesis, a pyruvate kinase and 3-phosphoglycerate mutase deletion mutant was constructed. It could be shown that pyruvate kinase is essential for growth both on the non PTS (phosphotransferase system) sugar ribose and on the gluconeogenetic substrates acetate and citrate. Global gene expression analyses and enzyme assays with a suppressor mutant showed, that the growth of the pyruvate kinase mutant on acetate and citrate could be restored by overexpression of the mez gene encoding the malic enzyme. Further it was shown, that the pyruvate kinase mutant accumulates glycolytic precursors for L-serine biosynthesis. To inactivate the 3-phosphoglycerate mutase, the functional expressed gene was identified by sequence analysis and growth experiments. A pgm deletion mutant was only able to grow in presence of both a glycolytic and a gluconeogenetic substrate. Thereby, the non-PTS sugar maltose has been metabolised referentially to glucose that is taken up via a PTS system. It has been shown that the mutant accumulates the unphosphorylated L-serine precursor glycerate. To investigate the L-serine degradation the L-serine dehydratase encoding gene was identified in the C. glutamicum genome by sequence comparison. The subsequent deletion of the sdaA gene resulted in a 1.8-fold decreased L-serine degradation rate in comparison to the wild type but the overexpression of sdaA led to a 2-fold increased degradation rate. Interestingly, the overexpression of sdaA enabled C. glutamicum to grow on L-serine. The deletion of the sdaA gene in a C. glutamicum strain overexpressing the L-serine biosynthesis genes revealed a 960-fold gain of the L-serine accumulation. After an additional deletion of either the 3-phosphoglycerate mutase or pyruvate kinase encoding gene, a further increase of the L-serine formation was found. Further it was shown that the deletion of sdaA and the simultaneous reduction of the serine hydroxymethyltransferase activity, another L-serine converting enzyme, in a strain overexpressing the L-serine biosynthesis genes, led to a tremendous increase of L-serine accumulation up to 100 mM.
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